Artificial chromosomes comprising EHV sequences

ABSTRACT

The invention belongs to the field of animal health, in particular equine diseases caused by equine herpesvirus (EHV). The invention relates to artificial chromosomes comprising the genome of equine herpesviruses, methods of producing attenuated or virulent EHV with or without the insertion of foreign genes, EHV obtainable with said methods and pharmaceutical compositions comprising said viruses.

The invention belongs to the field of animal health, in particularequine diseases caused by equine herpesvirus (EHV). The inventionrelates to artificial chromosomes comprising the genome of equineherpesviruses, methods of producing attenuated EHV viruses, EHV virusesobtainable with said methods and pharmaceutical compositions comprisingsaid viruses.

BACKGROUND OF THE INVENTION

Equine herpesvirus 1 (EHV-1), a member of the Alphaherpesvirinae, is themajor cause of virus-induced abortion in equids and causes respiratoryand neurological disease. The entire DNA sequence of the EHV-1 strainAb4p has been determined (Telford, E. A. R. et al., 1992). Only fewgenes and gene products have been characterized for their relevance forthe virulence or immunogenicity of EHV-1 because the production of viralmutants is still relying on the generation of recombinant viruses byhomologous recombination between the viral genome and respective foreignDNA to be inserted in cultured mammalian cells.

For control of EHV-1 infections, two different approaches are followed.First, modified live vaccines (MLVs) have been developed, including thestrain RacH (Mayr, A. et al., 1968; Hübert, P. H. et al., 1996), whichis widely used in Europe and the United States. Second, inactivatedvaccines and independently expressed viral glycoproteins have beenassessed for their immunogenic and protective potential. Among theglycoproteins that were expressed using recombinant baculoviruses arethe glycoproteins (g) B, C, D, and H, which induced partial protectionagainst subsequent challenge EHV-1 infection in a murine model (Awan, A.R. et al:, 1990; Tewari, D. et al., 1994; Osterrieder, N. et al., 1995;Stokes, A. et al., 1996). However, the use of MLVs has advantages overkilled and subunit vaccines. MLVs are highly efficient in inducingcell-mediated immune responses, which are most likely to be responsiblefor protection against disease (Allen, G. P. et al., 1995; Mumford, J.A. et al., 1995).

Herpesvirus glycoproteins are crucially involved in the early stages ofinfection, in the release of virions from cells, and in the directcell-to-cell spread of virions by fusion of neighboring cells. To date,11 herpes simplex virus type 1 (HSV-1)-encoded glycoproteins have beenidentified and have been designated gB, gC, gD, gE, gG, gH, gI, gJ, gK,gL, and gM. HSV-1 mutants lacking gC, gE, gG, gI, gJ, and gM are viable,indicating that these genes are dispensable for replication in culturedcells. Comparison of the HSV-1 and equine herpesvirus 1 nucleotidesequences revealed that all of the known HSV-1 glycoproteins areconserved in EHV-1. According to the current nomenclature, theseglycoproteins are designated by the names of their HSV-1 homologs. Inaddition, a further envelope protein named gp1/2 and a tegument protein,the VP13/14 homolog of HSV-1, have been described to be glycosylated incase of EHV-1 (reviewed in Osterrieder et al., 1998). It is known thatEHV-1 gC, gE gI, and gM are not essential for growth in cell culture,whereas gB and gD appear to be essential for virus growth in culturedcells. The contributions of other EHV-1 glycoproteins to replication incultured cells are not known (Neubauer et al., 1997; Flowers et al.,1992).

The gp1/2 glycoprotein is encoded by gene 71 (Wellington et al., 1996;Telford et al., 1992) and was also shown to be nonessential for virusgrowth in vitro (Sun et al., 1996). In addition, a viral mutant carryinga lacZ insertion in the gene 71 open reading frame was apathogenic in amurine model of infection but still able to prevent against subsequentchallenge infection (Sun et al., 1996; Marahall et al. 1997). Inaddition, the KyA strain of EHV-1 harbors a major deletion in the codingsequences of gene 71 (Colle et al., 1996).

The technical problem underlying this invention was to provide a newtool and procedure to generate attenuated equine herpesviruses ofdefined specificity.

SUMMARY OF THE INVENTION

The above-captioned technical problem is solved by the embodimentscharacterized in the claims and the description.

The invention relates to artificial chromosomes comprising the genome ofEHV, methods of producing attenuated EHV, EHV obtainable with saidmethods and pharmaceutical compositions comprising said viruses.

FIGURE LEGENDS

FIG. 1:

Cloning strategy for introduction of mini F plasmid sequences into theRacH genome (A). PCR amplification of fragments bordering gene 71located in the US region of the genome (B) was done and the resultingBamHI-KpnI and SalI-SphI fragments were consecutively cloned into vectorpTZ18R(C). Mini F plasmid sequences were released from recombinantplasmid pHA2 (Adler et al., 2000) with PacI and cloned to give rise torecombinant plasmid p71-pHA2 (D). This plasmid was co-transfected withRacH DNA into RK13 cells and fluorescing virus progeny was selected.Viral DNA from green fluorescing virus progeny was used to transformEscherichia coli DH10B cells from which infectious RacH-BAC wasisolated. Restriction enzyme sites and scales (in kbp) are given.

FIG. 2:

Restriction enzyme digests of RacH and RacH-BAC. After separation by0.8% agarose gel electrophoresis, fragments were transferred to a nylonmembrane (Pharmacia-Amersham) and hybridized with a labelled pHA2 probe(see FIG. 1). Reactive fragments which are present due to insertion ofmini F plasmid sequences are indicated by asterisks. Molecular weightmarker is the 1 kb ladder (Gibco-BRL). The restriction enzymes used areindicated.

FIG. 3:

Plaque sizes of RacH and RacH-BAC. Plaque sizes were determined on RK13cells by measuring diameters of 150 plaques each. Plaque sizes of RacHwere set to 100%, respectively, and plaque sizes of virus progenyreconstituted from BAC were compared to those of the parental virus.Standard deviations are given.

FIG. 4:

Principle of the deletion of the genes encoding for gD (a) or gM (b) inRacH-BAC by replacing the open reading frames with the kanamycinresistance gene (kan^(R)) using E/T cloning. The kan^(R) gene wasamplified by PCR using the primers listed in Table 1, and the ampliconwas electroporated into DH10B cells containing RacH-BAC and plasmidpGETrec which expresses the enzymes necessary for E/T cloning afterarabinose induction (Schumacher et al., 2000). Kanamycin-resistantcolonies were picked, DNA was isolated and subjected to Southern blotanalysis using a kan^(R)-specific probe. In both gD-negative RacH-BAC(c) and gM-negative RacH-BAC (d), fragments of the expected sizes (gD:20.4 kbp; gM: 9.3 kbp specifically reacted with the kan^(R) probe.

DETAILED DESCRIPTION OF THE INVENTION

Before the embodiments of the present invention it must be noted that asused herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a virus” includes aplurality of such viruses, reference to the “cell” is a reference to oneor more cells and equivalents thereof known to those skilled in the art,and so forth. Unless defined otherwise, all technical and scientificterms used herein have the same meanings as commonly understood by oneof ordinary skill in the art to which this invention belongs. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods, devices, and materials are now described. Allpublications mentioned herein are incorporated herein by reference forthe purpose of describing and disclosing the cell lines, vectors, andmethodologies which are reported in the publications which might be usedin connection with the invention. Nothing herein is to be construed asan admission that the invention is not entitled to antedate suchdisclosure by virtue of prior invention.

The invention relates to an artificial chromosome vector characterizedin that it comprises essentially the entire genome of an EHV strain fromwhich infectious progeny can be reconstituted after transfection into apermissive cell.

With the artificial chromosome vectors according to the presentinvention, safe EHV-vaccines comprising EHV with defined attenuationscan be generated. Such viruses are useful for the preparation of a safelive vaccine for use in the prevention and/or treatment of EHVinfections (see infra). The invention provides the possibility for afast and efficient manipulation of the EHV genome which remains fullyinfectious for eukaryotic cells or is modified into areplication-deficient virus. There was a long lasting need in the artfor such a tool to handle and manipulate the huge genome of EHV. Lastly,the EHV nucleic acid can be used as a polynucleotide vaccine which isapplied either topically or systemically to naive or primed horses andmay also be applied in utero.

The present invention is illustrated in example 1 showing the cloning ofthe entire genome of EHV-1 as an infectious mini F plasmid (‘bacterialartificial chromosome’, BAC) into Escherichia coli. The generation ofsaid BAC was not trivial and was posed many difficulties, including thepreparation and extraction of sufficient amounts of circular DNA. Thecircularized form of recombinant viral DNA was needed to transform DH10Bcells with the recombinant DNA in order to prepare the mini Fplasmid-cloned EHV DNA. To obtain sufficient amounts of circular viralDNA, early viral transcription was blocked by the addition of 100 μg perml of cycloheximide after infection of cells. Viral DNA was thenprepared and used for transformation of DH10B cells. Only from cellstreated with cycloheximide was it possible to extract sufficient amountsof circular DNA and to obtain DH10B clones containing the entire RacHgenome.

“Essentially” means that the EHV genome is complete with the exceptionthat it may carry a mutation as set out infra.

“Artificial chromosome” relates to any known artificial chromosomes,such as yeast, or preferably bacterial artificial chromosomes.

Preferably, a bacterial artificial chromosome (BAC) according to theinvention is a vector used to clone large DNA fragments (100- to 300-kbinsert size) in Escherichia coli cells which is based on naturallyoccurring F-factor plasmid found in the bacterium E. coli (Shizuya, H.,B. Birren, U. J. Kim et al. 1992. Cloning and stable maintenance of300-kilobase-pair fragments of human DNA in Escherichia coli using anF-factor-based vector. Proceedings National Academy of Science 89:8794-8797). The type of vector is preferably based on a F-plasmidreplicon containing the origin of replication (oriS) and its own DNApolymerase (repE) as well as the genes parA and parB involved inmaintaining its copy number at a level of one or two per E. coli. Theantibiotic resistance marker is preferably Cm-resistance.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV is EHV-1.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV is EHV-4.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain isRacH.

According the invention, any type of mutation can be introduced into theEHV genome, in order to obtain a replication-deficient and/or attenuatedEHV virus. Such mutations include, but are not limited to any mutation(e.g. deletion, insertion, substitution) relating to the glycoproteinsgB, gC, gD, gE, gG, gI, gJ, gL and gM, gp1/2 and any combinationthereof. Preferably, said mutations are deletion mutations, i.e. therespective glycoproteins such as e.g. gM are completely deleted.

Thus, the invention preferably relates to an artificial chromosomevector according to the invention, characterized in that the EHV strainis lacking the glycoprotein gB.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gC.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gD.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gE.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gG.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gH.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gI.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gK.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gL.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gM.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the EHV strain islacking the glycoprotein gp1/2.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the artificialchromosome is a bacterial artificial chromosome (BAC). Said BAC's can bepropagated in any bacterium known to the skilled person, e.g. andpreferably Escherichia coli.

The invention preferably relates to an artificial chromosome vectoraccording to the invention, characterized in that the artificialchromosome is a yeast artificial chromosome (YAC).

The invention preferably relates to an artificial chromosome vectorRacH-BAC according to the invention, characterized in that theartificial chromosome as deposited under the accession number ECACC01032704 with the ECACC in Porton Down, UK (European Collection of CellCultures, CAMR, Salisbury, Wiltshire SP4 0JG, UK), on Mar. 27, 2001, byDr. N. Osterrieder (Bodden Blick 5A, Insel Riems, D-17498 Germany). Theviability of this deposit was tested and confirmed on Mar. 27, 2001, andis capable of reproduction.

Another important embodiment of the present invention is apolynucleotide vaccine encoding an an artificial chromosome vector orEHV contained therein according to the invention.

Yet another important embodiment of the present invention is the use ofan artificial chromosome vector according to the invention for thegeneration of infectious EHV.

The invention furthermore relates to a method for the generation of aninfectious EHV, characterized in that an artificial chromosome vectoraccording to the invention is used to infect a suitable cell line andthe shedded virus is collected and purified.

The invention furthermore relates to a method for the generation of anattenuated EHV, characterized in that the EHV sequence contained in anartificial chromosome vector according to the invention is specificallymodified by molecular biology techniques.

Said modifications may be carried out by methods known in the art, e.g.site directed mutagenesis see e.g. Sambrook et al. (1989) MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.

Furthermore, the invention relates to a EHV obtainable by a methodaccording to the invention.

Another very important embodiment is a pharmaceutical compositioncomprising a polynucleotide according to the invention and optionallypharmaceutically acceptable carriers and/or excipients. Such apolynucleotide according to the invention may also be used in apharmaceutical composition within the scope of this invention, e.g. forDNA vaccination.

One example of a targeted system of administration, e.g. forpolynucleotides according to the invention is a colloidal dispersionsystem. Colloidal dispersion systems comprise macromolecule complexes,nanocapsules, microspheres and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles and liposomes orliposome formulations. Liposomes are the preferred colloidal systemaccording to the invention. Liposomes are artificial membrane vesicleswhich are useful as carriers in vitro and in vivo. These formulationsmay carry a cationic, anionic or neutral charge. It has been shown thatlarge unilamellar vesicles (LUV) ranging from 0.2-4.0 μm in size mayenclose a major part of an aqueous buffer solution with largemacromolecules. RNA, DNA and intact virions can be encapsulated in theaqueous phase inside and transported to the target in a biologicallyactive form (Fraley R et al., 1981, Trends Biochem Sci 6, 77-80). Inaddition to mammalian cells, liposomes have also proved suitable for thetargeted transporting of nucleotides into plant, yeast and bacterialcells. In order to be an efficient gene transfer carrier the followingproperties should be present: (1) the genes should be enclosed with highefficiency without reducing their biological activity; (2) there shouldbe preferential and substantial binding to the target cell compared withnon-target cells; (3) the aqueous phase of the vehicle should betransferred highly efficiently into the target cell cytoplasm; and (4)the genetic information should be expressed accurately and efficiently(Mannino R. J. et al., 1988, BioTechniques 6, 682-690).

The composition of the liposomes usually consists of a combination ofphospholipids, particularly high phase transition temperaturephospholipids, e.g. combined with steroids such as cholesterol. Otherphospholipids or other lipids may also be used. The physicalcharacteristics of the liposomes depend on the pH, the ion concentrationand the presence of divalent cations.

The pharmaceutical composition according to the invention may alsocontain a vector according to the invention, e.g. a BAC vectorcomprising an EHV genome as described supra, as a naked “gene expressionvector”. This means that the vector according to the invention is notassociated with an adjuvant for targeted administration (e.g. liposomes,colloidal particles, etc.). A major advantage of naked DNA vectors isthe absence of any immune response caused by the vector itself.

The EHV nucleic acid can be used as a polynucleotide vaccine (seepharmaceutical composition, supra) which is applied either topically(e.g. intranasally) or systemically to naive or primed horses and mayalso be applied in utero.

Another very important embodiment is a pharmaceutical compositioncomprising an EHV virus according to the invention and pharmaceuticallyacceptable carriers and/or excipients. A pharmaceutically acceptablecarrier can contain physiologically acceptable compounds that act, forexample, to stabilize or to increase the absorption or form part of aslow release formulation of the EHV virus or the polynucleotideaccording to the invention. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers or excipients(see also e.g. Remington's Pharmaceutical Sciences (1990). 18th ed. MackPubl., Easton). One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition.

Furthermore, the invention relates to the use of a polynucleotideaccording to the invention in the manufacture of a vaccine for theprevention and/or treatment of EHV infections.

Furthermore, the invention relates to the use of an EHV virus accordingto the invention in the manufacture of a vaccine for the preventionand/or treatment of EHV infections.

Furthermore, the invention relates to the use of the BAC technology toestablish a highly virulent and genetically well characterized EHV whichcan be used for immunization and challenge studies for use e.g. invaccine potency studies.

Furthermore, the invention relates to the use of EHV BACs according tothe invention to generate mutant BACs that are generated taking intoaccount appearing genetic or antigenetic variants of EHV. This relatesto one or more mutations present withing, new variants' of EHV which canbe easily introduced in the existing EHV BAC.

The following example is intended to aid the understanding of theinvention and should in no way be regarded as limiting the scope of theinvention.

EXAMPLE 1

Construction of an EHV-1 Bacterial Artificial Chromosome

A genetically uniform population of RacH (256^(th) passage) wasisolated. With RacH, passage 257, Rk13 cells were infected and a motherpool was established. Virus of one additional passage on RK13 cells wasused to infect RK13 cells, from which viral DNA was prepared. Tenmicrograms (μg) of viral DNA were co-transfected with 10 μg of plasmidp71-pHA2 (FIG. 1) into RK13 cells. For construction of plasmid p71-pHA2,2.0 and 2.4 kbp fragments on either side of the EHV-1 gene 71 (FIG. 1;Table 1) were amplified by polymerase chain reaction (PCR) using primerscontaining appropriate restriction enzyme sites (Table 1). Bothfragments were subsequently cloned into pTZ18R (Pharmacia-Amersham) toobtain plasmid p71 (FIG. 1). A BAC vector (pHA2; Messerle et al., 1997)containing the Eco-gpt and GFP (green flourescent protein) genes underthe control of the HCMV (human cytomegalovirus) immediate early promoterwas released as a PacI fragment from plasmid pHA2 and inserted into thePacI sites of the 2.0 and 2.4 kbp fragment cloned in p71 (FIG. 1; Table1). Virus progeny was harvested and individual plaques expressing thegreen fluorescent protein (GFP) were isolated and subjected to threerounds of plaque purification until virus progeny stained homogenouslygreen under the fluorescent microscope (Seyboldt et al., 2000).Similarly, co-transfections of p71-pHA2 and DNA of EHV-1 strain KentuckyA (KyA) were performed and the recombinant virus was purified tohomogeneity. Recombinant virus DNA was prepared (Schumacher et al.,2000) and electroporated into Escherichia coli strain DH10B (Messerle etal., 1997; Schumacher et al., 2000). Electrocompetent bacteria wereprepared as described (Muyrers et al., 1999; Narayanan et al., 1999;Zhang et al., 1998) and electroporation was performed in 0.1 cm cuvettesat 1250 V, a resistance of 200 Ω, and a capacitance of 25 μF (Easyjectelectroporation system, Eurogenentec). Transformed bacteria wereincubated in 1 ml of Luria-Bertani (LB) medium (28) supplemented with0.4% glucose for 1 hr at 37° C., and then plated on LB agar containing30 μg/ml chloramphenicol. Single colonies were picked into liquid LBmedium, and small scale preparations of BAC DNA were performed byalkaline lysis of Escherichia coli (Schumacher et al., 2000). Largescale preparation of BAC DNA was achieved by silica-based affinitychromatography using commercially available kits (Qiagen, Macherey &Nagel).

From the chloramphenicol-resistant bacterial colonies, one colony eachwas chosen and named RacH-BAC which contained the EHV-1 RacH genome.RACH-BAC DNA was cleaved with restriction enzymes BamHI, EcoRI andHindIII and the restriction enzyme patterns were compared to those ofparental viral DNA. (Schumacher et al., 2000). The calculated andexpected changes in the banding pattern after insertion of the mini Fplasmid into the gene 71 locus were observed in RacH-BAC. In contrast,no other differences in restriction enzyme patterns as compared to theparental virus were obvious (FIG. 2). After purification of RacH-BAC DNAusing affinity chromatography, RK13 cells were transfected with 1 μg ofrecombinant DNA. At one day after transfection, foci of greenfluorescent cells were visible which developed into plaques on thefollowing days after infection (FIG. 3). From these results we concludedthat the RacH strain of EHV-1 was cloned as an infectious full-lengthviral DNA in Escherichia coli. Deletion of gene 71 in RacH-BAC resultedin a less than 10% reduction in plaque size (FIG. 3).

TABLE 1 Seq. ID Fragment or plasmid No. Primer Sequence generated 1.Gen71 1.Fr. 5′-GCA ggtaccTTTGCACAACTTTAGGATGAC-3′ 2.0-kb flank forp71-pHA2 for 2. Gen71 1.Fr. 5′-GAT ggatcc CTttaattaaGTAGACGCGGCTGTAGTAAC-3′ 2.0-kb flank for p71-pHA2 rev 3. Gen712.Fr. 5′-ACA gtcgac CT ttaattaaTCGGGGAACTACTCACACTC-3′ 2.4-kb flank forp71-pHA2 for 4. Gen71 2.Fr. 5′-CGA gcatgcAGTTTTACGCGAAGGATATAC-3′ 2.4-kbflank for p71-pHA2 rev 5. Kan950 for 5′-GCCAGTGTTACAACCAATTAACC-3′Kan^(r)950 gene 6. Kan950 rev 5′-CGATTTATTCAACAAAGCCACG-3′ Kan^(r)950gene 7. gM950EHV 5′-

Kan^(r)950 gene for gM for

GCCAGTGTTACAACCAATTAAC-3′ deletion 8. gM950EHV 5′-

Kan^(r)950 gene for gM rev

CGATTTATTCAACAAAGCCACG-3′ deletion 9. gD-950 for 5′-

Kan^(r)950 gene for gD

CGATTTATTCAACAAAGCCACG-3′ deletion 10. gD-950-1 rev 5′-

Kan^(r)950 gene for gD

GCCAGTGTTACAACAAATTAACC-3′ deletionMutagenesis of EHV-1 BACs

For mutagenesis of RacH-BAC DNA in Escherichia coli, recE- andrecT-catalyzed reactions promoting homologous recombination betweenlinear DNA fragments, also referred to as E/T cloning, was performed(Muyrers et al., 1999; Zhang et al., 1999). Plasmid pGETrec (kindlyprovided by Dr. Panos Ioannou, Murdoch Institute, Melbourne, Australia)harboring the recE, recT and bacteriophage λ gam gene (Narayanan et al.,1999) was transformed into RacH-BAC-containing DH10B cells. Afterinduction of recE, recT and gam by addition of 0.2% arabinose,electrocompetent cells were prepared essentially as described (Muyrerset al., 1999). To delete the gD and gM gene in RACH-BAC, the kanamycinresistance gene (kan^(R)) of plasmid pACYC177 (Stratagene) was amplifiedby PCR. The designed primers contained 50 nucleotide homology armsbordering the desired deletion within gD or gM and 20 nucleotides foramplification of kan^(R) (Table 1). The resulting 0.95 kbp fragment waspurified from an agarose gel (Qiagen) and electroporated intopGETrec-containing RacH-BAC cells. Colonies harboring the cam^(R) andkan^(R) genes were identified on plates containing both antibiotics.

H-BACΔgD and H-BACΔgM DNA were isolated from Escherichia coli bychromatography and subjected to restriction enzyme digestion andSouthern blot analysis (FIG. 4) transfection studies were performed.Whereas RacH-BAC and H-BACΔgM were able to induce viral plaques on RK13cells, H-BACΔgD was able to induce plaques on cells expressing gD intrans only. The gD cells transiently expressed EHV-1 gD aftertransfection of a recombinant plasmid in which gD is under control ofthe HCMV immediate early promoter/enhancer. These observations indicatedthat EHV-1 gD is essential for virus growth in vitro.

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1. A bacterial artificial chromosome vector as deposited under ECACCaccession No.
 01032704. 2. The bacterial artificial chromosome vectoraccording to claim 1, wherein said bacterial artificial chromosomevector as deposited under ECACC accession No. 01032704 further lacks asequence coding for glycoprotein gM.
 3. The bacterial artificialchromosome vector of claim 1, wherein a foreign sequence of anotherviral, bacterial or parasitic pathogen is added to said bacterialartificial chromosome vector as deposited under ECACC accession No.01032704.
 4. A polynucleotide encoding an artificial chromosome vector,which vector is characterized in that it comprises the entire genome ofan EHV strain deposited under ECACC accession No.
 01032704. 5. A methodfor generating EHV which comprises infecting a suitable cell line withthe artificial chromosome vector according to claim 1, allowing thevector to replicate and shed virus, collecting the shed virus andpurifying the collected virus.
 6. A method for generating an attenuatedEHV which comprises modifying by molecular biology techniques the EHVsequence contained in an artificial chromosome vector according toclaim
 1. 7. The method according to claim 1 wherein a foreign sequenceof another viral, bacterial or parasitic pathogen is added to theartificial chromosome vector.
 8. A method for generating a virulent EHVwhich comprises modifying by molecular biology techniques the EHVsequence contained in an artificial chromosome vector according toclaim
 1. 9. The method according to claim 8 wherein a foreign sequenceof another viral, bacterial or parasitic pathogen is added to theartificial chromosome vector.